Published online May 28, 2020. doi: 10.3748/wjg.v26.i20.2570
Peer-review started: January 1, 2020
First decision: January 19, 2020
Revised: March 2, 2020
Accepted: May 13, 2020
Article in press: May 13, 2020
Published online: May 28, 2020
Circulating microRNAs (miRNAs) are potential biomarkers for many diseases. However, they can originate from non-disease specific sources, such as blood cells, and compromise the investigations for miRNA biomarkers. While small extracellular vesicles (sEVs) have been suggested to provide a purer source of circulating miRNAs for biomarkers discovery, the most suitable blood sample for sEV miRNA biomarker studies has not been defined.
To compare the miRNA profiles between matched serum and plasma sEV preparations to determine their suitability for biomarker studies.
Matched serum and plasma samples were obtained from 10 healthy controls and 10 patients with esophageal adenocarcinoma. sEV isolates were prepared from serum and plasma using ExoQuickTM and quantified using NanoSight. RNA was extracted from sEV preparations with the miRNeasy Serum/Plasma kit and profiled using the Taqman Openarray qPCR. The overall miRNA content and the expression of specific miRNAs of reported vesicular and non-vesicular origins were compared between serum and plasma sEV preparations. The diagnostic performance of a previously identified multi-miRNA biomarker panel for esophageal adenocarcinoma was also compared.
The overall miRNA content was higher in plasma sEV preparations (480 miRNAs) and contained 97.5% of the miRNAs found in the serum sEV preparations (412 miRNAs).The expression of commonly expressed miRNAs was highly correlated (Spearman’s R = 0.87, P < 0.0001) between the plasma and serum sEV preparations, but was consistently higher in the plasma sEV preparations. Specific blood-cell miRNAs (hsa-miR-223-3p, hsa-miR-451a, miR-19b-3p, hsa-miR-17-5p, hsa-miR-30b-5p, hsa-miR-106a-5p, hsa-miR-150-5p and hsa-miR-92a-3p) were expressed at 2.7 to 9.6 fold higher levels in the plasma sEV preparations compared to serum sEV preparations (P < 0.05). In plasma sEV preparations, the percentage of protein-associated miRNAs expressed at relatively higher levels (Ct 20-25) was greater than serum sEV preparations (50% vs 31%). While the percentage of vesicle-associated miRNAs expressed at relatively higher levels was greater in the serum sEV preparations than plasma sEV preparations (70% vs 44%). A 5-miRNA biomarker panel produced a higher cross validated accuracy for discriminating patients with esophageal adenocarcinoma from healthy controls using serum sEV preparations compared with plasma sEV preparations (AUROC 0.80 vs 0.54, P < 0.05).
Although plasma sEV preparations contained more miRNAs than serum sEV preparations, they also contained more miRNAs from non-vesicle origins. Serum appears to be more suitable than plasma for sEV miRNAs biomarkers studies.
Core tip: Current evidence suggests that circulating small extracellular vesicles (sEVs) function as delivery cargo shuttles for various molecules. MicroRNAs are small non-coding RNAs with important roles in the regulation of gene expression, are often dysregulated in diseases, and are relatively stable in the circulation. MicroRNAs circulating in sEVs are consequently considered as highly suitable candidates for use as non-invasive biomarkers. Extracellular vesicle preparations derived from serum and plasma are recognised to be enriched in sEVs, but not purely comprised of them. Most circulating sEV microRNA biomarker studies have used plasma, but here we show that sEVs isolated from serum are less contaminated with blood cell and protein-associated microRNAs.